We have continued to focus on the role of insulin-like growth factors (IGF's) and tumor specific translocations in the pathogenesis of pediatric sarcomas, specifically rhabdomyosarcomas (RMS), Ewing's sarcomas/PNET (Ewing's family of tumors EFT), and osteogenic sarcomas (OS). We have recently completed a randomized study of GH-IGF-I inhibition using a somatostatin analog in dogs with osteosarcomas. We hypothesized that lowering IGF-I levels would lead to increased apoptosis in tumor cells treated with standard chemotherapy. Forty-six dogs were randomized on this study and the data are currently being analyzed. Preliminary analysis reveals that treatment with no the somatostatin analog led to a 50% decrease in circulating IGF-I levels and no increase in survival. Utilizing cDNA micro-array analysis, we have compared the expression profiles in 3T3 fibroblasts overexpressing IGF-I receptors following ligand stimulation to profiles in 3T3 cells overexpressing insulin receptors following ligand stimulation. In general, IGF-I receptor stimulation leads to more changes in growth and differentiation gene expression, while insulin receptor stimulation leads to changes in metabolic pathways. We found that the oncogene twist was upregulated by IGF-I receptor stimulation and this appears to help mediate the anti-apoptotic signaling of IGF-I. We have continued to utilize C2 mouse myoblasts overexpressing IGF2 to determine specific consequences of IGF2 overexpression in a muscle background. We have found that IGF-II induced resistance to apoptosis is mediated in part through a PI3K-AKT-p70S6K pathway with p70S6K being the most distal component. This is reversed by rapamycin, and we are exploring possible clinical applications of this drug in treatment of pediatric sarcomas with activated PI3K-AKT-p70S6K pathways. We are currently focusing on the role of FKHR in mediating IGF related anti-apoptotic events as well, since this gene is altered in the t(2;13) translocation in alveolar rhabdomyosarcoma and appears to be an important signal in this pathway. We are continuing to collaborate on a genetic model of rhabdomyosarcoma in mice, and have found that this model predicts abnormalities in the p16-CDK-4-cylcin D-Rb pathway in these tumors and have confirmed such abnormalities in human tumor specimens. We have used cDNA microarray analysis to identify genetic determinants of metastasis in an established mouse orthotopic model of osteosarcoma with spontaneous metastases. . One gene identified that appears to be highly correlated with metastatic behavior is ezrin, a member of the ezrin-radixin-moesin family of proteinsthat function as plasma membrane-cytoskeletal linkers. Preliminary functional data support the role of this protein in metastatic behavior in osteosarcoma.